The occurence of polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofurans (PCDF) in the environment and the workplace poses a serious potential threat to human health. These compounds are among the most toxic and teratogenic low molecular weight compounds known.
Chlorinated dibenzo-p-dioxins are formed from the condensation of two orthochlorophenates under conditions of high alkalinity, pressure and temperature. 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) is formed as a contaminant by the condensation of two trichlorophenate molecules in the production of the herbicide 2,4,5-T. Synthesis of polychlorinated biphenyls (PCBs) can also produce chlorinated dibenzofurans as contaminants and various chlorinated dibenzo-p-dioxin isomers are formed in the commercial production of pentachlorophenol.
Burning of organic compounds, under appropriate conditions of temperature of combustion, chemical nature and combustion chamber residence time can also lead to the production of PCDDs and PCDFs. This has been verified by the analysis of flue gas emission and fly ash of municipal incinerators in the Netherlands. This phenomenon has also been observed in laboratory models. The pyrolysis of organohalogens such as chlorinated phenols and PCBs leads to the formation of PCDDs and PCDFs.
The PCDDs consist of 75 isomers and congeners which vary in the number and position of the chlorines. One of the most toxic, TCDD is a nearly planar tricyclic aromatic molecule. It is very stable in soil, has few readily reactive groups and is very insoluble in water (6.times.10.sup.-10 M). The toxicity of these compounds has been shown to be dependent upon the number and location of the chlorines, with TCDD being the most toxic isomer, with an oral LD.sub.50 for guinea pigs of 1 .mu.g/kg. TCDD is considered to be one of the most potent low molecular weight molecule known, with a minimum lethal dose orders of magnitude below sodium cyanide, strychnine and curare. In contrast to these agents, which are fast-acting, a single dose of TCDD may result in death weeks later. Species susceptibility to TCDD varies, with the hamster being 5,000 times less sensitive than the guinea pig. The exact cause of death is unknown. Hepatic necrosis may be involved in rats, rabbits and mice, but not in guinea pigs.
Many of the studies leading to present knowledge of the toxicity associated with PCDDs and PCDFs were initiated in response to specific poisoning episodes. The occurence of these compounds as a contaminant in chlorophenols which were used in hide curing led to an outbreak of chick edema in this country in 1957. It was determined that contaminated fat removed from the hides was being used as chicken feed. Hydropericardium as well as kidney and liver damage of the poultry were described.
The effects of human exposures have been seen in a number of industrial processing plant incidents. An acne outbreak in Germany in 1954 was traced to trichlorophenol contaminated with PCDDs and PCDFs. Similar outbreaks of chloracne were noted in 2,4,5-T production plants in France, Germany and the United States in the 1950's and 1960's. In 1971 the use of waste oil contaminated with TCDD for dust control in a riding arena led to the death of a large number of horses and other small animals, as well as chloracne in children who played in the area. The contamination by dibenzofuran analogs of rice oil in Japan led to many cases of chloracne. The explosion of a chemical plant in Seveso, Italy in 1976 led to TCDD exposure and was thought to be responsible for the occurrence of chloracne in humans as well as a number of livestock losses. The use of Agent Orange (a mixture of n-butyl esters of 2,4-D and 2,4,5-T), a defoliant, in Vietnam between 1962 and 1969 led to the contamination of large areas by TCDD.
Animal studies have demonstrated the pleomorphic nature of TCDD toxicity. These include prolonged wasting syndrome prior to death, lymphoid involution and embryotoxicity and or teratogenicity, hyperkeratoses and chloracne, edema, hyperplasia of the epithelium of the stomach, intestines and urinary bladder, hepatocellular damage and thymic involution. Acneform lesions are the most common known toxic manifestation of TCDD exposure in humans. This response is thought to be caused by a thickening of the epidermis (acanthosis), hyperkeratosis and metaplastic change in the sebaceous glands to a squamous epithelium. Chloracne results as the keratinaceous material plugs hair follicles and the sebaceous glands become cystic.
Since the health hazards associated with these toxic compounds are of major public concern, a rapid, accurate and inexpensive means of detecting such compounds is of great importance. Unfortunately, a simple means of detecting these compounds has proven to be a long-standing and significant problem in the art.
In the past, it has been shown that TCDD and its congeners can induce aminolevulinic acid synthetase, which is the initial and rate limiting enzyme of heme synthesis which is correlated with the occurence of hepatic porphyria in animals, and aryl hydrocarbon hydroxylase (AHH) which is a cytochrome P-450 microsomal monoxygenase used for detoxification of xenobiotics and implicated in the activation of many procarcinogens to ultimate carcinogens. Further work using AHH responsive and non-responsive inbred strains of mice showed that the ability to express this enzyme induction is inherited as an autosomal dominant at the Ah locus with TCDD being about 30,000 times as potent as 3-methylcholanthrene. It was also postulated and later demonstrated that a TCDD receptor species exists and can be isolated in the cytosol fraction of mouse livers. This led to the development of a TCDD competitive binding procedure which allowed the affinity of TCDD binding with this receptor site to be compared to that of its isomers and congeners.
The in vivo induction of the hyperkeratinization response to TCDD exposure in an in vitro model system has been demonstrated by Knutson and Poland, Cell 22, 27-36 (1980). This hyperkeratinization response is thought to be responsible for the occurence of chloracne. It was found that TCDD did not produce an acute toxic response in 23 various cell cultures as judged by cell growth, viability and morphology. It was shown, however, that "XB" cells, derived as a cloned epithelial cell line from a mouse teratoma, in co-culture with irradiated 3T3 cells would exhibit a keratinization response to TCDD exposure when grown at high cell density. High density XB/3T3 cultures not treated with TCDD did not show this response.
Keratinization was induced in the XB/3T3 system at a concentration as low as 3.2 pg of TCDD per ml. This model is particularly relevant since it deals with an induced differentiation of the intact cell using an endpoint (hyperkeratinization) known to be relevant to human exposure, i.e. chloracne.
A correlation has been demonstrated between the degree of toxicity of TCDD isomers and congeners and the extent of enzyme induction, receptor binding and hyperkeratinization response. Certain chemical structural characteristics have been related to toxic potential and other measured parameters. These are as follows: (1) that potent inducers of these biological effects have halogen atoms located at three of the four lateral ring positions (2,3,7,8); (2) that at least one ring position is nonhalogenated; (3) the order of potency of halogenated substitution is Br&gt;Cl&gt;F; (4) the molecule must maintain a rigid planar geometry for maximal potency. This geometry must be tricyclic (furans, dioxins, azobenzenes, aniline, etc.) or bicyclic but bridged such as PCBs and biphenylenes. The configuration for potency must be a 3.ANG..times.10.ANG.rectangle and modulation of potency from inactive, through low level to high level is dependent on the positions halogenated.
The toxicological effects of these structural characteristics cannot be discerned by methods of chemical analysis alone but must be expressed in an intact biological system. There are 22 possible TCDD isomers alone and precise analysis requires their availability as standard as well as their separation by a high resolution analytical technique such as HPLC, gas chromatography and mass spectrometry, which techniques are not amenable for routine analytical screening.
A bioassay, which is based on an intact living system, is needed as a broad screen for the detection of the biological activity consistent with that of the most toxic dioxin congeners and isomers. Such an assay could be used to determine the relative potency of individual pure compounds or of the aggregate potency of mixtures of compounds as in complex enviornmental samples.
Attempts to develop an in vitro bioassay for dioxinlike activity have verified the induction of keratinization in the XB/3T3 cell system by TCDD and have further demonstrated its potential usefulness in the semiquantitative detection of this activity in extracts of environmental samples. See Gierthy and Crane, J. Toxicology and Applied Pharmacology, 74, 91-98 (1984). The relative potency of these samples correlated well with the relative contamination of the samples by PCDFs. However, keratinization induction is not useful in practice due to the instability of the trait in the XB target cells. Utilization of this trait in a simple in vitro bioassay is therefore impossible, due to the complications that arise from the necessity of initiating repeated new cell cultures.